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library("devtools")
Loading required package: usethis
Registered S3 method overwritten by 'htmlwidgets':
method from
print.htmlwidget tools:rstudio
install_github("jw156605/SLICER")
Skipping install of 'SLICER' from a github remote, the SHA1 (cb1be8ac) has not changed since last install.
Use `force = TRUE` to force installation
library(Seurat)
Registered S3 method overwritten by 'data.table':
method from
print.data.table
Attaching SeuratObject
library(readr)
library(tidyverse)
Registered S3 methods overwritten by 'dbplyr':
method from
print.tbl_lazy
print.tbl_sql
── Attaching packages ──────────────────────────────────────────────────────── tidyverse 1.3.2 ──
✔ ggplot2 3.4.0 ✔ dplyr 1.0.10
✔ tibble 3.1.8 ✔ stringr 1.5.0
✔ tidyr 1.2.1 ✔ forcats 0.5.2
✔ purrr 0.3.5
── Conflicts ─────────────────────────────────────────────────────────── tidyverse_conflicts() ──
✖ dplyr::filter() masks stats::filter()
✖ dplyr::lag() masks stats::lag()
library(HGC)
library(latticeExtra)
Loading required package: lattice
Attaching package: ‘latticeExtra’
The following object is masked from ‘package:ggplot2’:
layer
library(lattice)
library(RColorBrewer)
library(clusterProfiler)
clusterProfiler v4.4.4 For help: https://yulab-smu.top/biomedical-knowledge-mining-book/
If you use clusterProfiler in published research, please cite:
T Wu, E Hu, S Xu, M Chen, P Guo, Z Dai, T Feng, L Zhou, W Tang, L Zhan, X Fu, S Liu, X Bo, and G Yu. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. The Innovation. 2021, 2(3):100141
Attaching package: ‘clusterProfiler’
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library(org.Mm.eg.db)
Loading required package: AnnotationDbi
Loading required package: stats4
Loading required package: BiocGenerics
Attaching package: ‘BiocGenerics’
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duplicated, eval, evalq, Filter, Find, get, grep, grepl, intersect, is.unsorted,
lapply, Map, mapply, match, mget, order, paste, pmax, pmax.int, pmin, pmin.int,
Position, rank, rbind, Reduce, rownames, sapply, setdiff, sort, table, tapply,
union, unique, unsplit, which.max, which.min
Loading required package: Biobase
Welcome to Bioconductor
Vignettes contain introductory material; view with 'browseVignettes()'. To cite
Bioconductor, see 'citation("Biobase")', and for packages 'citation("pkgname")'.
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Loading required package: S4Vectors
Attaching package: ‘S4Vectors’
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Attaching package: ‘IRanges’
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Attaching package: ‘AnnotationDbi’
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library(AnnotationDbi)
exp = read_csv("normalized_data.csv")
New names:
• `` -> `...1`
• `` -> `...151`
• `` -> `...240`
• `` -> `...289`
• `` -> `...352`
• `` -> `...391`
• `` -> `...394`
• `` -> `...450`
• `` -> `...462`
Rows: 14414 Columns: 463
── Column specification ─────────────────────────────────────────────────────────────────────────
Delimiter: ","
chr (1): ...1
dbl (454): M1_A01, M1_A02, M1_A03, M1_A05, M1_A06, M1_A08, M1_A09, M1_A10, M1_B01, M1_B03, M1...
lgl (8): ...151, ...240, ...289, ...352, ...391, ...394, ...450, ...462
ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.
exp = discard(exp,~all(is.na(.) |. == ""))
exp = distinct(exp, `...1`, .keep_all = TRUE)
# filtering out the reprogramming transcription factors
exp = filter(exp, !`...1`%in% c("Mef2c", "Gata4", "Tbx5"))
exp = column_to_rownames(exp, var="...1")
seurat.obj = CreateSeuratObject(counts = exp)
seurat.obj = NormalizeData(seurat.obj)
Performing log-normalization
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seurat.obj = FindVariableFeatures(seurat.obj, selection.method = "vst", nfeatures = 10000)
Calculating gene variances
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all.genes = rownames(seurat.obj)
seurat.obj = ScaleData(seurat.obj, features = all.genes)
Centering and scaling data matrix
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seurat.obj = RunPCA(seurat.obj, features = VariableFeatures(seurat.obj))
PC_ 1
Positive: Ehd4, Acaa2, Dsp, Ldhb, Nid2, Ryr2, Myl4, Epb4.1, Smpx, Tnni3
Ttn, Tnnt2, Mgst3, Sort1, Ldb3, Jup, Mlf1, Baiap2l1, Rrad, Mgrn1
Fryl, Ppp1r3c, Fam134b, Fabp3, Cenpa, Usp28, Clu, Pcyt1a, Ddit4l, Cd24a
Negative: Fstl1, Bgn, Cd200, Col3a1, Rbp1, Tagln2, Fam198b, Postn, Col1a2, Tagln
Fbln2, Rbms3, Lox, Fbn1, Loxl1, Dhrs3, Marcks, Col8a1, 11-Sep, Col5a2
Clca1, Lrrc8a, Spry2, Anxa1, Ogn, Nars, Gstm2, Fbln5, Asns, Acta2
PC_ 2
Positive: Top2a, Prc1, Smc2, Ccnb1, Birc5, Cdk1, Ube2c, Rrm2, Mki67, Pbk
Ckap2l, Asf1b, Cks2, Tpx2, Cdca8, Kif20b, 2810417H13Rik, Cks1b, Anln, Tacc3
Spc25, Hmmr, Hmgb2, Shcbp1, Ndc80, Mad2l1, Nuf2, Cenpe, Ect2, Cdca2
Negative: Cst3, B2m, Sptbn1, Malat1, Ypel5, Itm2b, Gyg, Parp3, Tspyl1, Serpine2
Lama2, Igfbp7, Ogn, B4galt1, Gpx3, Ctsa, Asah1, Pdcd4, Itgb5, Fhl2
Pam, Col4a2, Lhfp, Tcn2, Mxra8, Smim14, Ctsd, Akap13, Prex2, Impact
PC_ 3
Positive: Cst3, Col4a1, Flna, Igfbp7, Col1a1, Col4a2, Myl9, Sorbs2, Fhl2, Itgb1
Tns1, Col14a1, Ankrd1, Tpm2, Lbh, Pcolce, Tuba1a, Postn, Tuba1b, Itga1
Col5a1, Actn4, Pdlim2, Myom1, Pdgfrb, Tbx20, Sorbs1, Acta2, Pcdh18, Macf1
Negative: Gpm6a, Mgst1, Hpgd, Gsta4, Ptgs1, Upk1b, Pkhd1l1, F11r, Msln, Bst1
Maf, Ezr, Mpzl2, Cxadr, Krt8, Cd55, Igfbp6, Igfbp4, Acsl4, Bex4
Gcnt1, Vnn1, Ftl1, Efna5, Slpi, Ptp4a2, Tmem108, Tmem154, Csde1, Bnc1
PC_ 4
Positive: S100a6, Tpm1, Tpm4, Actb, Nme1, Zyx, Lgals1, Tpm2, Cryab, Actg1
Wisp1, Gapdh, Gm12070, Actn1, Timp1, Slc25a5, Sec13, Eif5a, Psat1, Creb3l1
Tnfrsf12a, Ccnd2, Csf1, Pkm, Pcolce, Acta2, Des, Rcn3, Col1a1, Wdr1
Negative: Egfl7, Ctla2a, Gja4, Emcn, Cdh5, Notch1, Klhl4, Gimap6, Pecam1, Stab1
Gpr116, Lyl1, Tie1, Entpd1, Ctla2b, Fli1, Myct1, Sox7, Thsd1, Eltd1
Gngt2, Mmrn2, S1pr1, Mecom, Tgfbi, Klhl24, Tmem2, Apoe, Plxnd1, Akap13
PC_ 5
Positive: Egfl7, Notch1, Klhl4, Stab1, Cdh5, Emcn, Cgnl1, Pecam1, Gja4, Ramp2
Lyl1, Col4a1, Gpr116, Tie1, Entpd1, Zfp69, Loxl2, Eltd1, Plxnd1, Thsd1
Cxcl16, Col5a1, Tek, S1pr1, Txndc5, Tgfbi, Odc1, Eng, Sox7, Myct1
Negative: Spcs3, Impact, Ypel5, Cpne8, Lnp, Arhgap44, Sptbn1, Myo10, Nexn, Arid5b
Arl5a, Braf, Klhl9, Cpne3, Lama2, Mrfap1, Serping1, Mlf1, Zdhhc2, Mier3
Gm2a, Dstn, Emc2, Myoz2, Ncam1, Rhobtb3, Cryab, Gucy1a3, Ttn, Idh1
ElbowPlot(seurat.obj)
seurat.obj = FindNeighbors(seurat.obj, dims = 1:15)
Computing nearest neighbor graph
Computing SNN
seurat.obj = FindClusters(seurat.obj, resolution = 1.75)
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
Number of nodes: 454
Number of edges: 16805
Running Louvain algorithm...
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Maximum modularity in 10 random starts: 0.4797
Number of communities: 8
Elapsed time: 0 seconds
identified_cells = read.csv("paper_cell_states.csv", row.names = 1, header=TRUE)
identified_cells = identified_cells[match(rownames(seurat.obj@meta.data), rownames(identified_cells)),]
seurat.obj@meta.data$cc_paper = identified_cells$CC
clusters = DimPlot(seurat.obj, group.by = "RNA_snn_res.1.75", label = TRUE)
condition = DimPlot(seurat.obj, group.by = "cc_paper", label = TRUE)
condition|clusters
Idents(seurat.obj) = "RNA_snn_res.1.75"
seurat.obj = RunUMAP(seurat.obj, dims = 1:15)
02:10:43 UMAP embedding parameters a = 0.9922 b = 1.112
02:10:43 Read 454 rows and found 15 numeric columns
02:10:43 Using Annoy for neighbor search, n_neighbors = 30
02:10:43 Building Annoy index with metric = cosine, n_trees = 50
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02:10:43 Writing NN index file to temp file /var/folders/fj/09brpj8s5mz1c5wxktl80s080000gn/T//RtmpiZsIsa/file862e1f60cef0
02:10:43 Searching Annoy index using 1 thread, search_k = 3000
02:10:43 Annoy recall = 100%
02:10:44 Commencing smooth kNN distance calibration using 1 thread with target n_neighbors = 30
02:10:45 Initializing from normalized Laplacian + noise (using irlba)
02:10:45 Commencing optimization for 500 epochs, with 17028 positive edges
Using method 'umap'
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02:10:46 Optimization finished
clusters = DimPlot(seurat.obj, group.by = "RNA_snn_res.1.75", label = TRUE, reduction = "umap")
condition = DimPlot(seurat.obj, group.by = "cc_paper", label = TRUE, reduction = "umap")
condition|clusters
seurat.obj = RunTSNE(seurat.obj, dims = 1:15)
DimPlot(seurat.obj, reduction = "tsne", group.by = 'RNA_snn_res.1.75')
markers = FindAllMarkers(seurat.obj, logfc.threshold = 0.25, min.pct = 0.1, only.pos = FALSE, test.use = 'MAST')
Calculating cluster 0
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Done!
Calculating cluster 1
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
Calculating cluster 2
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
Calculating cluster 3
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
Calculating cluster 4
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
Calculating cluster 5
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
Calculating cluster 6
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Combining coefficients and standard errors
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Done!
top_markers = markers %>%
group_by(cluster) %>%
slice_max(n = 2, order_by = avg_log2FC)
FeaturePlot(seurat.obj, features = c("Dhrs3"), min.cutoff = 'q10')
VlnPlot(seurat.obj, features = c("Rbp1", "Dhrs3"))
VlnPlot(seurat.obj, features = c("Fstl1", "Bgn"))
VlnPlot(seurat.obj, features = c("Serpinh1", "Dsp"))
VlnPlot(seurat.obj, features = c("Prc1", "Birc5"))
## cell cycle
s.genes <- str_to_title(cc.genes$s.genes)
g2m.genes <- str_to_title(cc.genes$g2m.genes)
seurat.obj = CellCycleScoring(seurat.obj, s.features = s.genes, g2m.features = g2m.genes)
clusters = DimPlot(seurat.obj, group.by = "cc", label = TRUE, reduction = "umap")
real_condition = DimPlot(seurat.obj, group.by = "cc_paper", label = TRUE, reduction = "umap")
clusters|real_condition
NA
NA
seurat_normalized = as.data.frame(GetAssayData(object = seurat.obj, slot = "data"))
seurat_normalized["Top2a", ]
seurat.sc = CreateSeuratObject(counts = exp)
seurat.sc = SCTransform(seurat.sc)
Calculating cell attributes from input UMI matrix: log_umi
Variance stabilizing transformation of count matrix of size 14028 by 454
Model formula is y ~ log_umi
Get Negative Binomial regression parameters per gene
Using 2000 genes, 454 cells
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Calculating gene attributes
Wall clock passed: Time difference of 5.798916 secs
Determine variable features
Place corrected count matrix in counts slot
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Set default assay to SCT
seurat.sc = RunPCA(seurat.sc, features = VariableFeatures(seurat.sc))
PC_ 1
Positive: Bgn, Fstl1, Postn, Col1a2, Cd200, Col3a1, Rbp1, Tagln, Dhrs3, Tagln2
Acta2, 11-Sep, Fbln2, Clca1, Lox, Wisp2, Col8a1, Anxa1, Fam198b, Marcks
Ogn, Col5a2, Fbn1, Pdpn, Asns, Adamts5, Acot2, Irgm1, Tcf21, Ptprb
Negative: Tnnt2, Nid2, Acaa2, Myl4, Ldhb, Dsp, Ehd4, Clu, Tnni3, Smpx
Ttn, Ryr2, Myl7, Cd24a, Gpx3, Epb4.1, Fam134b, Ppp1r3c, Sort1, Mgrn1
Mlf1, Rn45s, Ldb3, Cenpa, Rrad, Fabp3, Arhgap44, Mgst3, Lgals3, Ptrf
PC_ 2
Positive: Top2a, Smc2, Ccnb1, Cdk1, Prc1, Mki67, Rrm2, H2afz, Stmn1, Tpx2
Cdca8, Anln, Pbk, Birc5, Tacc3, Ckap2l, Asf1b, Mad2l1, Hmgn2, Hmmr
Ube2c, Rrm1, Cenpe, Kif20b, Cks2, Ndc80, Shcbp1, Spc25, Hmgb2, Cdca3
Negative: Cst3, Gyg, Malat1, Gpx3, Sptbn1, B2m, Ogn, Fhl2, Sparc, Dhrs3
Serpine2, Tnnt2, Impact, Rbp1, Col5a2, Sat1, Fn1, Itgb5, B4galt1, Col4a2
Serpina3h, Lama2, Tmsb4x, Postn, Gucy1a3, Adamtsl2, Hsd11b1, Adamts5, Ttyh2, Myl9
PC_ 3
Positive: Mgst1, Pdpn, Vcam1, Ftl1, F11r, Efna5, Cyp1b1, Sdc4, Ptgs1, Gpm6a
Steap1, Ezr, Upk1b, Ctsl, Dcn, Ccl2, Gsta4, Maf, Flrt2, Msln
Efemp1, Timp3, Igfbp4, Cxadr, Hpgd, Dpp4, Gja1, Mpzl2, F3, Bnc1
Negative: Myl9, Ankrd1, Acta2, Cst3, Sorbs2, Tnnt2, Fhl2, Mustn1, Tmsb4x, Flna
Pdlim3, Lmcd1, Tagln, Col4a1, Pls3, Lpp, Postn, Wisp2, Gyg, Gpx3
Sh3bgrl, Myl4, Col4a2, Itga8, Pdlim2, Fn1, Nid2, Vim, Myom1, Csrp2
PC_ 4
Positive: Tpm1, Acta2, Tagln, Anxa1, Actg1, Actb, Cav1, Anxa2, Myl9, Tmsb4x
Prss23, Efemp1, Cryab, Mustn1, Zyx, Flna, Arl5a, Lox, S100a4, Col8a1
Spcs3, Thbs1, Pls3, Fhl1, Ccnd2, Upk1b, Lpp, Map6, Plec, Ankrd1
Negative: Dcn, Abcg1, Rbp1, B2m, Igf2r, Hsd11b1, Gpx3, B4galt1, Mmp2, Mgp
Serpine2, Cxcl1, Serpina3g, Cp, Angptl2, Ttyh2, Serpina3h, Rab31, Ccl7, Serpina3n
Tmem176b, Srpx2, Gpm6b, Sepp1, Ctsl, Btg2, Ccl2, Btg1, Dhrs3, Casp4
PC_ 5
Positive: Serpina3h, Col1a2, Col1a1, Serpina3i, Col4a1, Lsp1, Csf1, Ch25h, Asns, Des
Ccl7, Wisp1, Col6a3, Adamtsl2, Gfpt2, Lgals1, Timp1, Serpina3g, Psat1, Ankrd1
Col4a2, Nars, Flnb, Pcolce, Postn, Chst11, Ccnd1, Ckb, Rn45s, Flna
Negative: Spcs3, Sptbn1, Btg1, Impact, Phactr2, Akap13, Rab31, Cd93, Cfh, Slc38a2
Myo10, Arl5a, Sh3bgrl, Adamts5, Ddah1, Tspan12, Fryl, Gucy1a3, Lmcd1, Canx
Id3, Adamtsl3, Prss23, Eln, Clca1, B230120H23Rik, 5430417L22Rik, Wisp2, AI597468, Fn1
identified_cells = identified_cells[match(rownames(seurat.sc@meta.data), rownames(identified_cells)),]
seurat.sc@meta.data$cc_paper = identified_cells$CC
seurat.sc = RunUMAP(seurat.sc, dims = 1:15)
seurat.sc = FindNeighbors(seurat.sc, dims = 1:15)
seurat.sc = FindClusters(seurat.sc, resolution = 1.75)
seurat.sc = CellCycleScoring(seurat.sc, s.features = s.genes, g2m.features = g2m.genes)
clusters = DimPlot(seurat.sc, group.by = "SCT_snn_res.1.75", label = TRUE, reduction = "umap")
real_condition = DimPlot(seurat.sc, group.by = "cc_paper", label = TRUE, reduction = "umap")
phase = DimPlot(seurat.sc, group.by = "Phase", label = TRUE, reduction = "umap")
clusters|real_condition|phase